The invention is directed to modified carbon, silicon, and germanium surfaces (including modified surfaces of alloys containing any combination of carbon, silicon and/or germanium). Specifically, the invention is directed to unoxidized carbon, silicon, and germanium surfaces modified to have bonded thereto a linker moiety capable of anchoring various other chemical moieties to the unoxidized surface. These modified surfaces are suitable for the fabrication of surface-bound chemicals, including surfaces of bound nucleic acids and nucleic acid arrays comprising two or more such surfaces.
Surfaces suitable for the immobilization of nucleic acids have become an increasingly important biological tool in recent years. Arrays of nucleic acid molecules, either as double-stranded segments or as short, single-stranded oligonucleotides, have been utilized for drug development, genetic sequencing, medical diagnostics, nucleic acid-ligand binding studies and DNA computing.1-21 The principal advantages of using surface-bound oligonucleotides over those in solution include ease of purification, conservation of material and reagents, reduction of interference between oligonucleotides, and facilitated sample handling.12 Previously explored surfaces for immobilization of nucleic acids include latex beads,5 polystyrene,1 carbon electrodes,22-25 gold,17,23,26-28 and oxidized silicon or glass.3,8,11,29-32 
The surface chemistries involved with these substrates, however, do not possess many of the desired characteristics of an ideal surface. These ideal surface characteristics include: surface flatness and homogeneity; control of surface properties; thermal and chemical stability; reproducibility; and amenability to nucleic acid immobilization and biochemical manipulation. More recently, the desire to use immobilized nucleic acid as a biosensor in integrated circuits33 would require an ideal surface to be amenable to integration into a microelectronics format. Thus, the limitations of the prior art surfaces and attachment chemistries indicate a need for alternative substrates that more closely resemble the ideal. As described herein, unoxidized crystalline carbon, silicon, and germanium, allotropes thereof, and alloys thereof offer advantages as substrates for immobilization of nucleic acids and other chemicals having similar or analogous reactivity because of their high purity, highly organized and defined crystalline structure and robustness. Silicon is particularly preferred due to its ubiquitous use in the microelectronics industry.
However, native silicon surfaces, for example, react with air under ambient conditions to form a thin surface layer of silicon oxide. This oxidized silicon surface is chemically similar to glass and suffers from some of the same drawbacks, namely inhomogeneity and variability in the relative number of Sixe2x80x94Oxe2x80x94Si and Sixe2x80x94OH linkages. This inhomogeneity and the concomitant chemical variability it engenders can lead to difficulties in the reproducibility and homogeneity of the subsequent chemical-modified surfaces, particularly as the silane chemistry generally employed to couple to such surfaces is itself prone to stability problems and difficult-to-control polymerization processes.34,35 
Chemical pathways for direct functionalization of silicon substrates without an oxide layer have opened up new possibilities for highly-controlled nucleic acid attachment. These new attachment methods provide modified silicon surfaces through direct carbon-silicon bonds,19,36-42 and have resulted in methyl-, chlorine-, ester-, or acid-terminated substrates.36,39,40,43,44 Although Wagner et al.19 describe using an N-hydroxysuccinimidyl ester-terminated silicon surface to immobilize a 1752 bp double-stranded section of DNA, this reference does not disclose a systematic, reproducible method to produce chemically-modified silicon surfaces in general, nor nucleic acid-modified silicon surfaces in particular.
Reactions of xcfx89-alkenes with silicon surfaces have been described in the prior art, either mediated by diacyl peroxides,36 through UV irradiation,19,41 or by direct thermal activation.39 The xcfx89-alkenes used have contained a variety of functional groups, including esters, acids,39,44 and chlorides.36 
The invention is directed to a method of creating modified carbon, silicon, and germanium surfaces comprising reacting a straight, branched, or cyclic alkene having a substitutent thereon with an unoxidized carbon, silicon, or germanium substrate to yield a modified substrate having bonded directly thereto, in the absence of any intervening oxygen atoms, substituted alkyl moieties.
A preferred embodiment of the invention is directed to a method of creating modified surfaces on unoxidized carbon, silicon, and germanium surfaces, the method comprising first reacting an amino-, carboxy, or thiol-modified and protected alkene with an unoxidized carbon, silicon, or germanium substrate to yield a surface of protected, amino-, carboxy, or thiol-modified alkane molecules covalently bonded to the substrate in the absence of any intervening oxygen molecules. Then the modified alkane molecules of the first step are deprotected, thereby yielding a surface of unprotected modified alkane molecules bonded to the substrate. Next, the deprotected modified alkane molecules are reacted with a crosslinker, whereby the crosslinker is attached to the modified alkane molecules. Lastly, molecules such as nucleic acids or proteins are attached to the crosslinker of the previous step, thereby yielding a surface of molecules immobilized on the substrate.
The invention is also directed to modified substrate produced according to these methods.
Similarly, another embodiment of the invention is drawn to a modified carbon, silicon or germanium substrate comprising a substrate selected from the group consisting of unoxidized, hydrogen-terminated carbon, silicon, and germanium and a layer of substituted alkane molecules bonded directly to the substrate, in the absence of any intervening oxygen molecules, the substituted alkane molecules having attached thereto a substituent selected from the group consisting of an amino-containing substituent, a carboxy-containing substituent, and a thiol-containing substituent.
In the preferred embodiment of the method, UV radiation mediates the reaction of a protected xcfx89-unsaturated aminoalkene, hydroxyalkene, carboxyalkene, or thioalkene (the most preferred being 10-aminodec-1-ene protected with t-BOC) with hydrogen-terminated silicon (001). Removal of the protecting group yields an aminoalkane-miodified, hydroxyalkane-modified, carboxyalkane, or thioalkane-modified silicon surface. These reactive terminal groups (amino, hydroxy, carboxy, thio) can then be coupled to modified oligonucleotides or other biomolecules using a heterobifunctional crosslinker, preferably SSMCC, thereby permitting the preparation of biomolecule arrays on an otherwise inert substrate.
A primary advantage of the present invention is that it enables the formation of highly ordered biomolecule arrays of defined surface density, including oligonucleotide arrays, on an inert, inorganic, unoxidized substrate.
Another distinct advantage of the present invention is that the surface density of biomolecules in the array can easily be controlled and manipulated. Control of surface density is provided in two fashions: In the first approach, binary mixtures of the protected xcfx89-unsaturated aminoalkene and dodecene are utilized in an initial UV-mediated coupling reaction. Here, a linear relationship was found between the mole fraction of xcfx89-unsaturated aminoalkene in the binary mixture and the density of oligonucleotide hybridization sites. In the second approach, only a portion of the protecting groups is removed from the surface by limiting the time allowed for the deprotection reaction, thereby limiting the number of available binding sites for oligonucleotide attachment.
Another advantage of the invention is the stability of the arrays. Oligonucleotide-modified surfaces formed utilizing the present invention are extremely stable and perform well in DNA hybridization assays. These surfaces provide an alternative to gold or glass for surface immobilization of oligonucleotides in DNA arrays as well as a route for the coupling of biomolecular recognition elements to semiconductor materials.
By permitting control of the density of the biomolecules within the array, the invention described herein also permits control of the hydrophobicity and concomitant wetting characteristics of the functionalized silicon surfaces, an important aspect in preparing reproducible and well-defined biomolecule arrays.